Uracil- and Pyridine-Containing HDAC Inhibitors Displayed Cytotoxicity in Colorectal and Glioblastoma Cancer Stem Cells.

Cancer stem cells (CSCs) are a niche of highly tumorigenic cells featuring self-renewal, activation of pluripotency genes, multidrug resistance, and ability to cause cancer relapse. Seven HDACi (1-7), showing either hydroxamate or 2'-aminoanilide function, were tested in colorectal cancer (CRC) and glioblastoma multiforme (GBM) CSCs to determine their effects on cell proliferation, H3 acetylation levels and in-cell HDAC activity. Two uracil-based hydroxamates, 5 and 6, which differ in substitution at C5 and C6 positions of the pyrimidine ring, exhibited the greatest cytotoxicity in GBM (5) and CRC (6) CSCs, followed by the pyridine-hydroxamate 2, with 2- to 6-fold higher potency than the positive control SAHA. Finally, increased H3 acetylation as well as HDAC inhibition directly in cells by selected 2'-aminoanilide 4 and hydroxamate 5 confirmed target engagement. Further investigation will be conducted into the broad-spectrum anticancer properties of the most potent derivatives and their effects in combination with approved, conventional anticancer drugs.

Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery.

RGD-cryptophycin and isoDGR-cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin-binding antimitotic agent across lysosomally cleavable Val-Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin αvß3, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21-L with different expression levels of integrin αvß3, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin-based SMDCs. However, no significant correlation between the in vitro biological activity of the conjugates and the integrin αvß3 expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective αvß3 integrin-mediated drug delivery.

Octreotide Conjugates for Tumor Targeting and Imaging.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvß3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)⁻cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvß3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

Blocking the association of HDAC4 with MAP1S accelerates autophagy clearance of mutant Huntingtin.

Autophagy controls and executes the turnover of abnormally aggregated proteins. MAP1S interacts with the autophagy marker LC3 and positively regulates autophagy flux. HDAC4 associates with the aggregation-prone mutant huntingtin protein (mHTT) that causes Huntington's disease, and colocalizes with it in cytosolic inclusions. It was suggested HDAC4 interacts with MAP1S in a yeast two-hybrid screening. Here, we found that MAP1S interacts with HDAC4 via a HDAC4-binding domain (HBD). HDAC4 destabilizes MAP1S, suppresses autophagy flux and promotes the accumulation of mHTT aggregates. This occurs by an increase in the deacetylation of the acetylated MAP1S. Either suppression of HDAC4 with siRNA or overexpression of the MAP1S HBD leads to stabilization of MAP1S, activation of autophagy flux and clearance of mHTT aggregates. Therefore, specific interruption of the HDAC4-MAP1S interaction with short peptides or small molecules to enhance autophagy flux may relieve the toxicity of mHTT associated with Huntington's disease and improve symptoms of HD patients.

Skin equivalents: a tool for the discovery and validation of pharmacodynamic biomarkers.

In the development of targeted oncology drugs, it is important to assess drug effectiveness in individual patients. We evaluated the possibility of reproducing in an ex-vivo system the biological effects observed in vitro and in vivo by the combined administration of two chemotherapeutic drugs, gemcitabine and a small inhibitor of Wee1. We found that modulation of both CDC2 phosphorylation and of a previously-identified gene signature was detectable in human skin equivalents obtained with primary keratinocytes from three individuals. Therefore, we suggest that human skin equivalents could represent a promising tool for the identification and validation of novel pharmacodynamic biomarkers.

Discovery of salermide-related sirtuin inhibitors: binding mode studies and antiproliferative effects in cancer cells including cancer stem cells.

Chemical changes performed on 1a (sirtinol) led to a series of SIRT1/2 inhibitors, in some cases more potent than 1a mainly against SIRT1. Tested in human leukemia U937 cells, the benzamide and anilide derivatives 1b, 1c, 2b, and 2c as well as the 4-(2-phenylpropyl)thioanalogue 4c showed huge apoptosis induction, while some sulfinyl and sulfonyl derivatives (5b, 5c, and 6a-c) were highly efficient in granulocytic differentiation. When assayed in human leukemia MOLT4 as well as in human breast MDA-MB-231 and colon RKO cancer cell lines, the anilide 2b (salermide) and the phenylpropylthio analogue 4b emerged as the most potent antiproliferative agents. Tested on colorectal carcinoma and glioblastoma multiforme cancer stem cells (CSCs) from patients, 2b was particularly potent against colorectal carcinoma CSCs, while 4b, 6a, and the SIRT2-selective inhibitor AGK-2 showed the highest effect against glioblastoma multiforme CSCs. Such compounds will be further explored for their broad-spectrum anticancer properties.

Benzodeazaoxaflavins as sirtuin inhibitors with antiproliferative properties in cancer stem cells.

Inhibition of sirtuins has recently been proposed as a promising anticancer strategy. Some of the new benzodeazaoxaflavins (2a, 2b, and 2d) here reported as SIRT1/2 inhibitors were endowed with pro-apoptotic properties in human U937 leukemia cells and, most importantly, together with the prototype MC2141 (1) displayed antiproliferative effects in cancer stem cells from patients with colorectal carcinoma and glioblastoma multiforme, known to be highly tumorigenic, resistant to conventional cancer chemotherapy, and responsible, at least in part, for cancer relapse or recurrence.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

Synthesis and biological activity of cyclotetrapeptide analogues of the natural HDAC inhibitor FR235222.

In the course of our ongoing efforts to discover new and more effective HDAC inhibitors useful for the development of promising anticancer candidates, we have recently undertaken a molecular modelling study on a small collection of FR235222 analogues, synthesized by us in the frame of a structure-activity relationship investigation, made in order to identify the key structural elements essential for the activity. Progress made in structure elucidation of HDAC active site, together with accurate docking calculations, provided new structural insights useful for a further refinement of the tetrapeptide scaffold which should assure an optimal interaction between the synthetic ligands and the biological target. Following the computer aided suggestions we synthesized six new cyclotetrapeptide analogues of the lead compound (3-8), bearing a carboxylic or an hydroxamic acid functionality as Zn binding moiety. Herein we describe their synthesis and their inhibition activity on different HDAC isoforms.

Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.

Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

Loss of histone deacetylase 4 causes segregation defects during mitosis of p53-deficient human tumor cells.

We investigated the role of histone deacetylase 4 (HDAC4) using RNA interference (RNAi) and knockout cells to specifically address its role in cell cycle progression in tumor and normal cells. Ablation of HDAC4 led to growth inhibition in human tumor cells but not to detectable effects in normal human dermal fibroblasts (NHDF) or myelopoietic progenitors. HDAC4-/+ or HDAC4-/- murine embryonic fibroblasts showed no detectable growth defects. On the other hand, HDAC4 RNAi in HeLa cells produced mitotic arrest followed by caspase-dependent apoptosis. Mitotically arrested cells showed chromosome segregation defects. Even though the growth of both p53-wild-type and p53-null tumor cells were affected by HDAC4 ablation, segregation defects were observed only in p53-null cells. HDAC4 associates with the PP2A-B56 regulatory subunit, which is known to be involved in chromosome segregation, and RNAi of either the structural subunit A or the regulatory subunit B56 of PP2A also caused chromosome segregation defects. We conclude that HDAC4 is required for cell cycle progression of tumor cells by multiple mechanisms, one of which seems to be specific to p53-deficient cells through chromosome segregation defects. On the contrary, HDAC4 is not required for the progression of NHDF. We therefore suggest that systemic selective interference with the expression or function of HDAC4 is expected to have a significant therapeutic window, in particular, for p53-deficient tumors.

Smoothened antagonists: a promising new class of antitumor agents.

BACKGROUND: Hedgehog signaling is essential for the development of most metazoans. In recent years, evidence has accumulated showing that many human tumors aberrantly re-activate this developmental signaling pathway and that interfering with it may provide a new strategy for the development of novel anti-cancer therapeutics. Smoothened is a G-protein coupled receptor-like protein that is essentially involved in hedgehog signal transduction and small molecule antagonists of Smoothened have started to show antitumor activity in preclinical models and in clinical trials. OBJECTIVE: We critically review the role of hedgehog signaling in normal development and in human malignancies, the available drug discovery tools and the classes of small molecule inhibitors that are in development. We further aim to address the potential impact that pathway antagonists may have on the treatment options of cancer patients. METHODS: Literature, patents and clinical trial results from the past 5 years were analyzed. CONCLUSIONS: 1) A large body of evidence suggests a frequent reactivation of hedgehog signaling in human cancer. 2) Smoothened is an attractive, highly druggable target with extensive preclinical and initial clinical validation in basal cell carcinoma. Several promising novel classes of Smoothened antagonists have been discovered and are being developed as anticancer agents. 3) Our knowledge of the biology of hedgehog signaling in cancer is still very incomplete and significant efforts will be required to understand how to use the emerging novel agents in the clinic.

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment.

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

Inhibition of class I histone deacetylase with an apicidin derivative prevents cardiac hypertrophy and failure.

AIMS: Recent studies have demonstrated the importance of chromatin remodelling via histone acetylation/deacetylation for the control of cardiac gene expression. Specific histone deacetylases (HDACs) can, in fact, play a positive or negative role in determining cardiac myocyte (CM) size. Here, we report on the effect on hypertrophy development of three inhibitors (HDACi) of class I HDACs. METHODS AND RESULTS: The compounds were first analysed in vitro by scoring hypertrophy, expression of foetal genes, and apoptosis of neonatal rat CMs stimulated with phenylephrine, an alpha1-adrenergic agonist. This initial screening indicated that a truncated derivative of apicidin with class I HDAC specificity, denoted API-D, had the highest efficacy to toxicity ratio, and was thus selected for further analysis in vivo. Administration of this drug significantly decreased myocardial hypertrophy and foetal gene expression after 1 week of pressure overload induced by thoracic aortic constriction (TAC) in mice. After 9 weeks of TAC, when manifest heart failure is encountered, mice treated with API-D presented with significantly improved echocardiographic and haemodynamic parameters of cardiac function when compared with untreated TAC-operated mice. CONCLUSION: The apicidin derivative, API-D, is capable of reducing hypertrophy and, consequently, the transition to heart failure in mice subjected to TAC. Treatment with this substance, therefore, holds promise as an important therapeutic option for heart failure.

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Probing the elusive catalytic activity of vertebrate class IIa histone deacetylases.

It has been widely debated whether class IIa HDACs have catalytic deacetylase activity, and whether this plays any part in controlling gene expression. Herein, it has been demonstrated that class IIa HDACs isolated from mammalian cells are contaminated with other deacetylases, but can be prepared cleanly in Escherichia coli. These bacteria preparations have weak but measurable deacetylase activity. The low efficiency can be restored either by: mutation of an active site histidine to tyrosine, or by the use of a non-acetylated lysine substrate, allowing the development of assays to identify class IIa HDAC inhibitors.

PP2A regulates HDAC4 nuclear import.

Different signal-regulated serine/threonine kinases phosphorylate class II histone deacetylases (HDACs) to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme C alpha, A alpha, B/PR55 alpha. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid or RNA interference have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import.

Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling.

Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.